Sunday, July 6, 2014

Inflammasome immortality

Immune system has many
ways to detect the presence of virulence factors. One such system is an assembly of the super-complex called inflammasome. For its proper activity this
super-complex requires close association of several components:
NALP3, ASC and pro-Caspase 1. Depending on initial trigger, NALP3 can be
replaced with NLRP1, AIM2, NLRC4 in inflammasome super-complex. Once
formed, inflammasome cleaves pro-IL-1 or pro-IL-18 into mature active
proteins IL-1beta and IL-18.

Earlier studies have shown that inflammasome assembly and function occurs in the cytoplasm. Now, two new papers published in Nature Immunology reveal that
inflammasome continues to function as a secreted, extracellular
template and amplifies local IL-1 production and recruitment of
granulocytes.

I am going to discuss
the results of several principal experiments from these studies.

In the first paper (1), the
authors generated macrophage line expressing ASC molecules fused to
fluorescent protein (ASC-FP). In resting state, ASC-FP were evenly
distributed in cytoplasm. Upon inflammasome activation, however,
ASC-FP formed oligomers. Interestingly, the authors detected
substantial amount of ASC-FP extracellularly. This release of ASC was
associated with cell death and requires caspase 1 activity. Treatment
of LPS-primed ASC-KO macrophage cytosol with recombinant ASC showed
that recombinant ASC had pro-caspase-1 and pro-IL-1 processing activity,
while LPS+ATP combination did not. These results indicated that
exogenously added ASC formed super-complexes with endogenous NALP3
and interacted with pro-caspase 1.

To directly observe this
phenomenon, the authors used red fluorescent tagged ASC-mCherry
construct. They found that (a) ASC-mCherry was phagocytosed and
released inside macrophage's cytosol after lysosomal membrane
destabilization and it formed oligomers, (b) as a result, an exogenous
ASC-mCherry acted as a danger signal for LPS-primed macrophages
inducing IL-1beta secretion, similar to silica or nigericin.

More importantly, using
two different fluorescent tagged ASC recombinant proteins, ASC-mCherry (red) and ASC-mCerulean (blue), the authors observed that
exogenously added ASC attracted endogenous ASC in super-complexes,
acted as a template for further oligomerization.

Finally, the authors
conducted several in vivo experiments with recombinant ASC and found
that injection of exogenous ASC oligomers induced local inflammatory
granulocytes recruitment independent of NALP3, but IL-1 receptor
dependent manner (this effect was enhanced in presence of anti-ASC
antibody or in presence of serum derived from autoimmune mouse
strain). Of note, there is opposite results regarding exogenous ASC ability to induce IL-1beta secretion from ASC-KO macrophages (compare Fig .3f and Fig. 3g versus Fig 5e). Either there is reporting error or in vitro bone marrow derived macrophages behave differently from ex vivo inflammatory macrophages.

In second paper (2), the
authors also confirmed that inflammasome components were detected in
supernatant within 15 min of macrophage activation and they formed
oligomers. They also found that addition of recombinant ASC oligomers
to LPS-primed ASC-KO macrophage cell line induced IL-1beta secretion
(however this required presence of ATP or NALP3, a slightly different
result from the first paper). The difference could be due to use
of primary macrophages versus macrophage cell line (or immortalized
macrophages) in different series of experiments, am obvious
limitation. Indeed, comparison of IL-1beta secretion results in Fig.
3J versus Fig. 4B, clearly indicates that dependency on ATP or NALP3
is a function of difference in cell types or difference in dose of
recombinant ASC oligomers.

In summary, these papers
showed that during inflammasome induced pyroptosis, dying cells
release active complexes of inflammasomes that are able to directly
process extracellular pro-IL-1 locally or alternatively these oligomers internalized by local macrophages and act as danger signals in
propagating inflammation. Since inflammasome-related mutations are
linked to several sterile inflammatory diseases, improved understanding of inflammasome biology could yield more targeted
therapeutics.